Methods for generating recombined polynucleotides

ABSTRACT

A method for in vitro construction of a library of recombined homologous polynucleotides from a number of different starting DNA templates and primers by induced template shifts during an polynucleotide synthesis is described, whereby 
     A. extended primers are synthesized by 
     a) denaturing the DNA templates 
     b) annealing primers to the templates, 
     c) extending the said primers by use of a polymerase, 
     d) stop the synthesis, and 
     e) separate the extended primers from the templates, 
     B. a template shift is induced by 
     a) isolating the extended primers from the templates and repeating steps A.b) to A.e) using the extended primers as both primers and templates, or 
     b) repeating steps A.b) to A.e), 
     C. this process is terminated after an appropriate number of cycles of process steps A. and B.a), A. and B.b), or combinations thereof. 
     Optionally the polynucleotides are amplified in a standard PCR reaction with specific primers to selectively amplify homologous polynucleotides of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplications 0307/97 filed Mar. 18, 1997, 0434/97 filed Apr. 17, 1997,0625/97 filed May 30, 1997, and U.S. Provisional applications Ser. No.60/044,836 filed Apr. 25, 1997 and 60/053,012 filed Jun. 24, 1997 thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to optimizing DNA sequences in order to(a) improve the properties of a protein of interest by artificialgeneration of genetic diversity of a gene encoding the protein ofinterest by the use of the so-called gene- or DNA shuffling technique tocreate a large library of "genes", expressing said library of genes in asuitable expression system and screening the expressed proteins inrespect of specific characteristics to determine such proteinsexhibiting desired properties or (b) improve the properties ofregulatory elements such as promoters or terminators by generation of alibrary of these elements, transforming suitable hosts therewith inoperable conjunction with a structural gene, expressing said structuralgene and screening for desirable properties in the regulatory element.

BACKGROUND OF THE INVENTION

It is generally found that a protein performing a certain bioactivityexhibits a certain variation between genera and even between members ofthe same species differences may exist. This variation is of course evenmore outspoken at the genomic level.

This natural genetic diversity among genes coding for proteins havingbasically the same bioactivity has been generated in Nature overbillions of years and reflects a natural optimization of the proteinscoded for in respect of the environment of the organism in question.

In today's society the conditions of life are vastly removed from thenatural environment and it has been found that the naturally occurringbioactive molecules are not optimized for the various uses to which theyare put by mankind, especially when they are used for industrialpurposes.

It has therefore been of interest to industry to identify such bioactiveproteins that exhibit optimal properties in respect of the use to whichit is intended.

This has for many years been done by screening of natural sources, or byuse of mutagenesis. For instance, within the technical field of enzymesfor use in e.g. detergents, the washing and/or dishwashing performanceof e.g. naturally occurring proteases, lipases, amylases and cellulaseshave been improved significantly, by in vitro modifications of theenzymes.

In most cases these improvements have been obtained by site-directedmutagenesis resulting in substitution, deletion or insertion of specificamino acid residues which have been chosen either on the basis of theirtype or on the basis of their location in the secondary or tertiarystructure of the mature enzyme (see for instance U.S. Pat. No.4,518,584).

In this manner the preparation of novel polypeptide variants andmutants, such as novel modified enzymes with altered characteristics,e.g. specific activity, substrate specificity, thermal, pH and saltstability, pH-optimum, pI, K_(m), V_(max) etc., has successfully beenperformed to obtain polypeptides with improved properties.

For instance, within the technical field of enzymes the washing and/ordishwashing performance of e.g. proteases, lipases, amylases andcellulases have been improved significantly.

An alternative general approach for modifying proteins and enzymes hasbeen based on random mutagenesis, for instance, as disclosed in U.S.Pat. No. 4,894,331 and WO 93/01285

As it is a cumbersome and time consuming process to obtain polypeptidevariants or mutants with improved functional properties a fewalternative methods for rapid preparation of modified polypeptides havebeen suggested.

Weber et al., (1983), Nucleic Acids Research, vol. 11, 5661, describes amethod for modifying genes by in vivo recombination between twohomologous genes. A linear DNA sequence comprising a plasmid vectorflanked by a DNA sequence encoding alpha-1 human interferon in the5'-end and a DNA sequence encoding alpha-2 human interferon in the3'-end is constructed and transfected into a rec A positive strain of E.coli. Recombinants were identified and isolated using a resistancemarker.

Pompon et al., (1989), Gene 83, p. 15-24, describes a method forshuffling gene domains of mammalian cytochrome P-450 by in vivorecombination of partially homologous sequences in Saccharomycescerevisiae by transforming Saccharomyces cerevisiae with a linearizedplasmid with filled-in ends, and a DNA fragment being partiallyhomologous to the ends of said plasmid.

In WO 97/07205 a method is described whereby polypeptide variants areprepared by shuffling different nucleotide sequences of homologous DNAsequences by in vivo recombination using plasmidic DNA as template.

U.S. Pat. No. 5,093,257 (Assignee: Genencor Int. Inc.) discloses amethod for producing hybrid polypeptides by in vivo recombination.Hybrid DNA sequences are produced by forming a circular vectorcomprising a replication sequence, a first DNA sequence encoding theamino-terminal portion of the hybrid polypeptide, a second DNA sequenceencoding the carboxy-terminal portion of said hybrid polypeptide. Thecircular vector is transformed into a rec positive microorganism inwhich the circular vector is amplified. This results in recombination ofsaid circular vector mediated by the naturally occurring recombinationmechanism of the rec positive microorganism, which include prokaryotessuch as Bacillus and E. coli, and eukaryotes such as Saccharomycescerevisiae.

One method for the shuffling of homologous DNA sequences has beendescribed by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci. USA, Vol.91, 10747-10751; Stemmer, (1994), Nature, vol. 370, 389-391). The methodconcerns shuffling homologous DNA sequences by using in vitro PCRtechniques. Positive recombinant genes containing shuffled DNA sequencesare selected from a DNA library based on the improved function of theexpressed proteins.

The above method is also described in WO 95/22625. WO 95/22625 relatesto a method for shuffling of homologous DNA sequences. An important stepin the method described in WO 95/22625 is to cleave the homologoustemplate double-stranded polynucleotide into random fragments of adesired size followed by homologously reassembling of the fragments intofull-length genes.

A disadvantage inherent to the method of WO 95/22625 is, however, thatthe diversity generated through that method is limited due to the use ofhomologous gene sequences (as defined in WO 95/22625).

Another disadvantage in the method of WO 95/22625 lies in the productionof the random fragments by the cleavage of the template double-strandedpolynucleotide.

A further reference of interest is WO 95/17413 describing a method ofgene or DNA shuffling by recombination of specific DNAsequences--so-called design elements (DE)--either by recombination ofsynthesized double-stranded fragments or recombination of PCR generatedsequences to produce so-called functional elements (FE) comprising atleast two of the design elements. According to the method described inWO 95/17413 the recombination has to be performed among design elementsthat have DNA sequences with sufficient sequence homology to enablehybridization of the different sequences to be recombined.

WO 95/17413 therefore also entails the disadvantage that the diversitygenerated is relatively limited. Furthermore the method described istime consuming, expensive, and not suited for automatisation.

Despite the existence of the above methods there is still a need forbetter iterative in vitro recombination methods for preparing novelpolypeptide variants. Such methods should also be capable of beingperformed in small volumes, and amenable to automatisation.

SUMMARY OF THE INVENTION

The present invention concerns briefly the utilization of template shiftof a newly synthesized DNA strand during in vitro DNA synthesis in orderto achieve DNA shuffling. By using this technique it is possible toobtain such results in a more expedient manner, and to some extent evena greater variation than in the above mentioned methods.

The method of the invention is also very well suited for adaption toautomatisation.

In a preferred embodiment the technique is used in combination with anerror-prone polymerase thereby introducing an even greater variation inthe library created.

More specifically the present invention relates to a method for theconstruction of a library of recombined homologous polynucleotides froma number of different starting single or double stranded parental DNAtemplates and primers by induced template shifts during an in vitropolynucleotide synthesis using a polymerase, whereby

A. extended primers or polynucleotides are synthesized by

a) denaturing parental double stranded DNA templates to produce singlestranded templates,

b) annealing said primers to the single stranded DNA templates,

c) extending said primers by initiating synthesis by use of saidpolymerase,

d) cause arrest of the synthesis, and

e) denaturing the double strand to separate the extended primers fromthe templates,

B. a template shift is induced by

a) isolating the newly synthesized single stranded extended primers fromthe templates and repeating steps A.b) to A.e) using said extendedprimers produced in (A) as both primers and templates, or

b) repeating steps A.b) to A.e),

C. the above process is terminated after an appropriate number of cyclesof process steps A. and B.a), A. and B.b), or combinations thereof, and

D. optionally the produced polynucleotides are amplified in a standardPCR reaction with specific primers to selectively amplify homologouspolynucleotides of interest.

In specific embodiments various modifications can be made in the processof the invention. For example it is advantageous to apply a defectivepolymerase either an error-prone polymerase to introduce mutations incomparison to the templates, or a polymerase that will discontinue thepolynucleotide synthesis prematurely to effect the arrest of thereaction.

Further modifications will be described below.

In a further aspect the invention relates to a method of identifyingpolypeptides exhibiting improved properties in comparison to naturallyoccurring polypeptides of the same bioactivity, whereby a library ofrecombined homologous polynucleotides produced by the above process arecloned into an appropriate vector, said vector is then transformed intoa suitable host system, to be expressed into the correspondingpolypeptides and displayed, said polypeptides are then screened in asuitable assay, and positive results selected.

In a still further aspect the invention relates to a method forproducing a polypeptide of interest as identified in the precedingprocess, whereby a vector comprising a polynucleotide encoding saidpolypeptide is transformed into a suitable host, said host is grown toexpress said polypeptide, and the polypeptide recovered and purified.

Finally, in further final aspects the invention relates to arecombined/shuffled protein, which is obtainable by any of the methodsaccording to the invention, and which is a recombined/shuffled proteincomprising the sequences disclosed herein (vide infra).

In those final aspects of the invention, the term "obtainable" denotesthat said protein is preferable obtained by a method according to theinvention. However a prior art known recombination/shuffling techniquesuch as those described in WO 95/22625 or WO 95/17413 may be used too,either alone or in combination with a method according to the invention,in order to obtain said recombined protein.

Accordingly, further final aspect of the invention are;

a recombined/shuffled protease obtainable by any of the methodsaccording to the invention, and comprising a recombined sequence, whichat least contain two different partial sequences from at least twodifferent wild-type proteases;

a recombined/shuffled lipase obtainable by any of the methods accordingto the invention, and comprising a recombined sequence, which at leastcontain two different partial wild-type sequences

a recombined/shuffled Pseudomonas lipases obtainable by any of themethods according to the invention, and comprising a recombinedsequence, which at least contain two different partial sequences from atleast two of the different wild-type Pseudomonas lipases;

a recombined/shuffled xylanase obtainable by any of the methodsaccording to the invention, and comprising a recombined sequence, whichat least contain two different partial sequences from at least twodifferent wild-type xylanases;

a recombined/shuffled cellulase obtainable by any of the methodsaccording to the invention, and comprising a recombined sequence, whichat least contain two different partial sequences from at least twodifferent wild-type cellulases;

a recombined/shuffled amylase obtainable by any of the methods accordingto the invention, and comprising a recombined sequence, which at leastcontain two different partial sequences from at least two differentwild-type amylases

a recombined/shuffled laccase obtainable by any of the methods accordingto the invention, and comprising a recombined sequence, which at leastcontain two different partial sequences from at least two differentwild-type laccases;

a recombined/shuffled phytase obtainable by any of the methods accordingto the invention, and comprising a recombined sequence, which at leastcontain two different partial sequences from at least two differentwild-type phytases.

Definitions

Prior to discussing this invention in further detail, the followingterms will first be defined.

"Shuffling": The term "shuffling" means recombination of nucleotidesequence fragment(s) between two or more homologous polynucleotidesresulting in output polynucleotides (i.e. polynucleotides having beensubjected to a shuffling cycle) having a number of nucleotide fragmentsexchanged, in comparison to the input polynucleotides (i.e. startingpoint homologous polynucleotides).

"Homology of DNA sequences or polynucleotides" In the present contextthe degree of DNA sequence homology is determined as the degree ofidentity between two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art, such as GAP provided in theGCG program package (Program Manual for the Wisconsin Package, Version8, August 1994, Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453).

"Homologous": The term "homologous" means that one single-strandednucleic acid sequence may hybridize to a complementary single-strandednucleic acid sequence. The degree of hybridization may depend on anumber of factors including the amount of identity between the sequencesand the hybridization conditions such as temperature and saltconcentration as discussed later (vide infra).

Using the computer program GAP (vide supra) with the following settingsfor DNA sequence comparison: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, it is in the present context believed that twoDNA sequences will be able to hybridize (using low stringencyhybridization conditions as defined below) if they mutually exhibit adegree of identity preferably of at least 70%, more preferably at least80%, and even more preferably at least 85%.

"heterologous": If two or more DNA sequences mutually exhibit a degreeof identity which is less than above specified, they are in the presentcontext said to be "heterologous".

"Hybridization:" Suitable experimental conditions for determining if twoor more DNA sequences of interest do hybridize or not is herein definedas hybridization at low stringency as described in detail below.

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using a x-ray film or a phosphoimager.

"primer": The term "primer" used herein especially in connection with aPCR reaction is an oligonucleotide (especially a "PCR-primer") definedand constructed according to general standard specifications known inthe art ("PCR A practical approach" IRL Press, (1991)).

"A primer directed to a sequence:" The term "a primer directed to asequence" means that the primer (preferably to be used in a PCRreaction) is constructed to exhibit at least 80% degree of sequenceidentity to the sequence fragment of interest, more preferably at least90% degree of sequence identity to the sequence fragment of interest,which said primer consequently is "directed to". The primer is designedto specifically anneal at the sequence fragment or region it is directedtowards at a given temperature. Especially identity at the 3' end of theprimer is essential.

"Flanking" The term "flanking" used herein in connection with DNAsequences comprised in a PCR-fragment means the outermost partialsequences of the PCR-fragment, both in the 5 and 3' ends of the PCRfragment.

"Polypeptide" Polymers of amino acids sometimes referred to as proteins.The sequence of amino acids determines the folded conformation that thepolypeptide assumes, and this in turn determines biological propertiesand activity. Some polypeptides consist of a single polypeptide chain(monomeric), whereas other comprise several associated polypeptides(multimeric). All enzymes and antibodies are polypeptides.

"Enzyme" A protein capable of catalysing chemical reactions. Specifictypes of enzymes to be mentioned are such as amylases, proteases,carbohydrases, lipases, cellulases, oxidoreductases, esterases, etc. Ofspecific interest in relation to the present invention are enzymes usedin detergents, such as proteases, lipases, cellulases, amylases, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in its first aspect to a method for theconstruction of a library of recombined homologous polynucleotides froma number of different starting single or double stranded parental DNAtemplates and primers by induced template shifts during an in vitropolynucleotide synthesis using a polymerase, whereby

A. extended primers or polynucleotides are synthesized by

a) denaturing a parental double stranded DNA template to produce singlestranded templates,

b) annealing said primers to the single stranded DNA templates,

c) extending said primers by initiating synthesis by use of saidpolymerase,

d) cause arrest of the synthesis, and

e) denaturing the double strand to separate the extended primers fromthe templates,

B. a template shift is induced by

a) isolating the newly synthesized single stranded extended primers fromthe templates and repeating steps A.b) to A.e) using said extendedprimers produced in (A) as both primers and templates, or

b) repeating steps A.b) to A.e),

C. the above process is terminated after an appropriate number of cyclesof process steps A. and B.a), A. and B.b), or combinations thereof, and

D. optionally the produced polynucleotides are amplified in a standardPCR reaction with specific primers to selectively amplify homologouspolynucleotides of interest.

According to the invention the polymerase may be a DNA or a RNApolymerase, specific polymerases to be mentioned are such as DNApolymerases like T4 polymerase, T7 polymerase, E. coli DNA polymerase Ior the Klenow fragment of DNA polymerase I, or thermostable polymerasessuch as Taq, Amplitaq, Vent, Pwo.

One of the advantages of the invention is that it makes it possible tocontrol the length of the extension of the primers in the reaction in aconvenient manner.

This can be accomplished by various means such as choice of polymerase,the physical and chemical conditions during the action of thepolymerase, e.g. pH, temperature, buffer, salt concentration, andaddition of various chemicals.

It is known that various polymerases carry out the DNA synthesis atdifferent rates (nucleotides incorporated pr. second). For example hasthe Klenow fragment of DNA polymerase I a limited extension ratecompared to e.g. the Taq polymerase (Sambrook et al. 1989).

Polymerases also display differences in processivity, which is theaverage number of nucleotides incorporated before the polymerasedissociates from the template/extended primer; again the Klenowpolymerase is an example of a polymerase with limited processivity.

The choice of polymerase is therefore an important means in controllingthe average extension of the primers.

These conditions may also exert an influence on the fidelity of thepolymerase (the rate by which point mutations are introduced; HIVreverse transcriptase is an example of a polymerase of low fidelity), aparameter useful in combining shuffling and mutagenesis.

In specific embodiments various modifications can be made in the processof the invention. For example it is advantageous to apply a defectivepolymerase either an error-prone polymerase to introduce mutations incomparison to the templates, or a polymerase that will discontinue thepolynucleotide synthesis prematurely to effect the arrest of thereaction. Such a defective polymerases that could be mentioned is aKlenow polymerase having low processivity.

In another embodiment of the invention polymerase will be added aftereach cycle, if the polymerase used is not thermostable.

According to the invention the starting single or double strandedparental templates may be different in that they contain different pointmutations in the same native polynucleotide (gene), or they can behomologous polynucleotides (genes) isolated from nature, which may beamplified by PCR, or they can be combinations thereof. The templatesused in the process of the invention are hereby homologous showing anidentity at the DNA level of e.g. more than 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, or even more than 50% identity.

It may be advantageous to use pre-selected templates comprisingmutations with improved properties of interest. The presentrecombination method of the invention will then recombine said improvedmutations for subsequent screening for even further improvements in theproperties of interest.

Said pre-selected templates with improved properties of interest mayhave been identified by standard procedures in the art comprising e.g.i) error-prone PCR of templates of interest followed by ii)screening/selection for templates with improved characteristic ofinterest. The mutagenesis frequency (low or high mutagenesis frequency)of the error-prone PCR step is preferably adjusted in relation to thesubsequent screening capacity, i.e. if the screening capacity is limitedthe error-prone PCR frequency is preferably low (i.e. one to twomutations in each template) (see WO 92/18645 for further details).

The arrest of the polymerase reaction in step A.d) may as indicatedabove be obtained in different ways, such as by raising the temperature,or adding specific reagents as described in WO 95/17413.

When raising the temperature for this purpose, it is preferred to usetemperatures between 90° C. and 99° C.

When using chemical agents DMSO is a possibility. Appropriate proceduresare mentioned in e.g. WO 95/17413.

The process of the invention uses annealing of the primers to thetemplates in step 1.b. In this context the annealing may be random orspecific, meaning either anywhere on the polynucleotide or at a specificposition depending on the nature of the primer.

Also, the primers to be used may be completely random primers(NNNNNNNNNNN) (N meaning a mixture of the four bases (A, T, G, C) isused at a particular position in the primer during synthesis),semi-random primers, or specific primers.

If the extended primers produced are to be separated from the primersduring the process it is convenient to use labeled templates in order toprovide a simple means for separation, a preferred marker is biotin ordigoxigenin.

According to the invention the number of cycles necessary will be lessthan 500, in most cases less than 200, and normally less than 100cycles.

In an embodiment of the invention the above in vitro shuffling iscombined with a subsequent in vivo shuffling by methods such as thosedescribed in WO 97/07205.

In its second aspect the invention relates to a method of identifyingpolypeptides exhibiting improved properties in comparison to naturallyoccurring polypeptides of the same bioactivity, whereby a library ofrecombined homologous polynucleotides produced by the above process arecloned into an appropriate vector, said vector is then transformed intoa suitable host system, to be expressed into the correspondingpolypeptides, said polypeptides are then screened in a suitable assay,and positive polypeptides selected.

In an embodiment it is contemplated that the polypeptides of interestencoded by the shuffled library are expressed as a suitable fusionprotein (e.g. as a hybrid with gIII of bacteriphage M13/fd) in order todisplay said recombined polypeptide on the surface of phage or bacteria.

In a third aspect the invention relates to a method for producing apolypeptide of interest as identified in the preceding process, wherebya vector comprising a polynucleotide encoding said polypeptide istransformed into a suitable host, said host is grown to express saidpolypeptide, and the polypeptide recovered and purified.

The use of partial random (semi-random) or completely random primers(mixtures of bases in a selected number or all positions in the primer)as initiation point for the DNA synthesis provide certain novelpossibilities for the combined use of shuffling and random mutagenesis.

It is often associated with difficulties to obtain an in vitrorecombination of polynucleotides that display relatively limitedhomology. By the use of an embodiment of the invention even very diversepolynucleotides can be forced to recombine.

According to that embodiment at least two templates (or two pools ofdiverse templates) are applied. The novel synthesis of the onepolynucleotide can then be based on only one strand (i.e. either thesense or the anti-sense strand), and the synthesis of the otherpolynucleotide is based the opposite strand.

This can be accomplished by isolating the complementary strands from thetwo templates, e.g. by having these strands labeled by biotin. Synthesisof DNA is initiated by annealing either specific, partly random orcompletely random primers to these templates and adding a suitablepolymerase. This can be performed as either separate reactions for thedifferent templates or in just one reaction. Synthesis should preferablybe performed under conditions that favor production of relatively shortnew fragments. These fragments can subsequently be isolated from thetemplates based on the affinity label. A PCR reaction is carried out onthese fragments and as the starting material originates from twodifferent strands, the newly synthesized fragments must recombine inorder to produce full length PCR products--a kind of forcedrecombination.

Also for this embodiment rapid PCR with short or no extension time canbe applied advantageously in order to enhance recombination, especiallyif pools of templates are used for the two strands.

The length of the primer and the annealing temperature utilized in theprocess determines if random primers will anneal and the number ofmismatches between the template and the primer that can be accommodated.By varying the primer length and the annealing temperature the method ofthe invention provides a means for achieving random mutagenesis within acertain nucleotide window representing the length of the primer. Themethod of the invention thereby provides substantial benefits comparedto other random mutagenesis approaches, especially the high probabilityfor several base substitutions close to each other in the primarysequence, e.g. the use of a completely random 20'mer (mixture of allfour nucleotides in all 20 positions) will according to theory undergiven experimental conditions give a certain reasonably high probabilityfor having several base substitutions close to each other.

Error prone PCR (=high mutagenesis frequency PCR) does not provide thispossibility. Error prone PCR provides a very low probability for havingmore than one base substitution within one codon (coding for one aminoacid in a translated polypeptide).

Obviously the substitution of only one base within a codon doesn'tprovide total random mutagenesis (at protein level) as only a limitedset of amino acid substitutions can be obtained by one base substitutionat DNA level (e.g. Methionine encoded by ATG-codon requires three basesubstitution to become the TGT or TGC-codon encoding Cysteine).

In one embodiment of the invention the process is therefore performed byusing random or semi-random primers having a length of from 6 to 200 bp,preferably from 10 to 70 bp, and better from 15 to 40 bp.

One of the advantages in the method of the invention is the robustness.In some embodiments the constant presence of full length templateprovides a further advantage avoiding PCR contamination problems.Furthermore it is much less laborius, with less hands on, than otherdescribed methods, thereby providing excellent possibilities forautomation.

PCR-primers

The PCR primers are constructed according to the standard descriptionsin the art. Normally they are 10-75 base-pairs (bp) long. However, forthe specific embodiment using random or semi-random primers the lengthmay be substantially longer as indicated above.

PCR-reactions

If not otherwise mentioned the PCR-reaction performed according to theinvention are performed according to standard protocols known in theart.

The term "Isolation of PCR fragment" is intended to cover as broad assimply an aliquot containing the PCR fragment. However preferably thePCR fragment is isolated to an extend which remove surplus of primers,nucleotides templates etc.

In an embodiment of the invention the DNA fragment(s) is(are) preparedunder conditions resulting in a low, medium or high random mutagenesisfrequency.

To obtain low mutagenesis frequency the DNA sequence(s) (comprising theDNA fragment(s)) may be prepared by a standard PCR amplification method(U.S. Pat. No. 4,683,202 or Saiki et al., (1988), Science 239, 487-491).

A medium or high mutagenesis frequency may be obtained by performing thePCR amplification under conditions which increase the misincorporationof nucleotides, for instance as described by Deshler, (1992), GATA 9(4),103-106; Leung et al., (1989), Technique, Vol. 1, No. 1, 11-15.

It is also contemplated according to the invention to combine the PCRamplification (i.e. according to this embodiment also DNA fragmentmutation) with a mutagenesis step using a suitable physical or chemicalmutagenizing agent, e.g., one which induces transitions, transversions,inversions, scrambling, deletions, and/or insertions.

Expressing the recombinant protein from the recombinant shuffledsequences

Expression of the recombinant protein encoded by the shuffled sequencein accordance with the second and third aspect of the present inventionmay be performed by use of standard expression vectors and correspondingexpression systems known in the art.

Screening and selection

In the context of the present invention the term "positive polypeptidevariants" means resulting polypeptide variants possessing functionalproperties which has been improved in comparison to the polypeptidesproducible from the corresponding input DNA sequences. Examples, of suchimproved properties can be as different as e.g. enhance or loweredbiological activity, increased wash performance, thermostability,oxidation stability, substrate specificity, antibiotic resistance etc.

Consequently, the screening method to be used for identifying positivevariants depend on which property of the polypeptide in question it isdesired to change, and in what direction the change is desired.

A number of suitable screening or selection systems to screen or selectfor a desired biological activity are described in the art. Examplesare:

Strauberg et al. (Biotechnology 13: 669-673 (1995) describes a screeningsystem for subtilisin variants having Calcium-independent stability;

Bryan et al. (Proteins 1:326-334 (1986)) describes a screening assay forproteases having a enhanced thermal stability; and

PCT-DK96/00322 describes a screening assay for lipases having improvedwash performance in washing detergents.

An embodiment of the invention comprises screening or selection ofrecombinant protein(s), wherein the desired biological activity isperformance in dish-wash or laundry detergents. Examples of suitabledish-wash or laundry detergents are disclosed in PCT-DK96/00322 and WO95/30011.

If, for instance, the polypeptide in question is an enzyme and thedesired improved functional property is the wash performance, thescreening may conveniently be performed by use of a filter assay basedon the following principle:

The recombination host cell is incubated on a suitable medium and undersuitable conditions for the enzyme to be secreted, the medium beingprovided with a double filter comprising a first protein-binding filterand on top of that a second filter exhibiting a low protein bindingcapability. The recombination host cell is located on the second filter.Subsequent to the incubation, the first filter comprising the enzymesecreted from the recombination host cell is separated from the secondfilter comprising said cells. The first filter is subjected to screeningfor the desired enzymatic activity and the corresponding microbialcolonies present on the second filter are identified.

The filter used for binding the enzymatic activity may be any proteinbinding filter e.g. nylon or nitrocellulose. The top-filter carrying thecolonies of the expression organism may be any filter that has no or lowaffinity for binding proteins e.g. cellulose acetate or Durapore®. Thefilter may be pre-treated with any of the conditions to be used forscreening or may be treated during the detection of enzymatic activity.

The enzymatic activity may be detected by a dye, fluorescence,precipitation, pH indicator, IR-absorbance or any other known techniquefor detection of enzymatic activity.

The detecting compound may be immobilized by any immobilizing agent e.g.agarose, agar, gelatin, polyacrylamide, starch, filter paper, cloth; orany combination of immobilizing agents.

If the improved functional property of the polypeptide is notsufficiently good after one cycle of shuffling, the polypeptide may besubjected to another cycle.

In an embodiment of the invention wherein homologous polynucleotidesrepresenting a number of mutations of the same gene is used as templatesat least one shuffling cycle is a back-crossing cycle with the initiallyused DNA fragment, which may be the wild-type DNA fragment. Thiseliminates non-essential mutations. Non-essential mutations may also beeliminated by using wild-type DNA fragments as the initially used inputDNA material.

Also contemplated to be within the invention is polypeptides havingbiological activity such as insulin, ACTH, glucagon, somatostatin,somatotropin, thymosin, parathyroid hormone, pituary hormones,somatomedin, erythropoietin, luteinizing hormone, chorionicgonadotropin, hypothalamic releasing factors, antidiuretic hormones,thyroid stimulating hormone, relaxin, interferon, thrombopoeitin (TPO)and prolactin.

A requirement to the starting parental DNA sequences, encoding thepolypeptide(s), to be shuffled, is that they are at least 50%, 60%, 70%,80%, 90%, or 95% homologous. DNA sequences being less homologous willhave less inclination to interact and recombine.

It is also contemplated according to the invention to shuffle parentalpolynucleotides that are homologous as indicated above originating fromwild type organisms of different genera.

Further, the starting parental templates to be shuffled may preferablyhave a length of from about 50 bp to 20 kb, preferably about 100 bp to10 kb, more preferred about 200 bp to 7 kb, especially about 400 bp to 2kb.

The starting parental DNA sequences may be any DNA sequences includingwild-type DNA sequences, DNA sequences encoding variants or mutants, ormodifications thereof, such as extended or elongated DNA sequences, andmay also be the outcome of DNA sequences having been subjected to one ormore cycles of shuffling (i.e. output DNA sequences) according to themethod of the invention or any other method (e.g. any of the methodsdescribed in the prior art section).

When using the method of the invention the resulting recombinedhomologous polynucleotides (i.e. shuffled DNA sequences), have had anumber of nucleotide fragments exchanged. This results in replacement ofat least one amino acid within the polypeptide variant, if comparing itwith the parent polypeptide. It is to be understood that also silentexchanges are contemplated (i.e. nucleotide exchange which does notresult in changes in the amino acid sequence).

MATERIALS AND METHODS

Specific Method Used in the Examples

A) DNA encoding different enzyme variants of the same gene or differentenzymes having the same type of activity encoded by homologous genes aremixed. The DNA is provided as either PCR fragments, plasmid, phage orgenomic DNA.

B) The resulting pool of DNA is mixed with DNA Polymerase, dNTP, asuitable buffer and primers (being either random oligomers (length of6-30 nucleotides) or specific oligomers (length of 6-50 nucleotides) ora combination of both types).

C) The PCR mixture is put into a PCR thermocycler (either cold or hot)in a suitable tube.

c1) The thermocycler is heated to a temperature of 90-100° C. for aperiod of time (typically 1-10 min) in order to denature the DNAtemplates.

c2) Thereafter the following procedure (cycle) is followed (repeated):The template is denatured (typically 90-100° C. for 0-5 minutes). Thenthe temperature is lowered (typically to a value between 10° C. and 90°C. for 0-5 minutes) to allow annealing of the primer to the singlestranded template. Now the temperature is raised again to denaturationtemperature (90-100° C.) allowing small extension of the primer to besynthesized by the DNA polymerase during ramping. Alternatively a shortextension period (typically 0-30 seconds at 70-75° C.) can be introducedto allow larger extensions of the primers to be generated. When thetemperature reaches a value where denaturation takes place, the extendedprimers and templates are again separated. This procedure can berepeated (typically between 1 to 99 cycles).

D) Having performed the desired number of cycles the generated small DNApolymers can be purified from the oligomers used as primers. One way isto isolate and clone a specific amplified band containing the genecoding for the polypeptide of interest into a suitable vector. This canbe done either on agarose gel (typically isolating fragments between 50to 1000 base pairs), by beads (using an affinity label on eithertemplates or primers) or through columns.

E) Then the purified (or the not purified) DNA polymers can be assembledin a standard PCR reaction (for instance 94° C., 5 minutes, (94° C., 30sec; 55° C., 30 sec; 72° C., 2 min)*25, 72° C. 5 minutes, 4° C.).

Specific primers or DNA polymers generated by specific primers can beadded in order to generate a specific DNA polymer containing the gene ofinterest. This As mentioned in point D, this DNA polymer can be purifiedand cloned into a vector of interest.

EXAMPLES Example 1

Method 1

The strong advantage of the method exemplified here is the robustnessand lack of PCR contamination problems, due to the constant presence ofparental template. Furthermore this method is less labor demanding thanmethods described in the prior art, thereby providing excellentpossibilities for automation.

Nine different plasmids containing DNA sequences encoding 9 differentvariants of the H. lanuginosa lipase gene, were mixed in equimolaramounts. The variant genes contained from two to seven mutationsscattered throughout the gene.

The DNA sequence of the H. lanuginosa lipase gene and the amino acidsequence of the lipase are disclosed in EP 0 305 216

The variants are indicated according to standard terminology asdescribed in e.g. EP 0 396 608, and WO 97/07202.

The following 9 variant genes were shuffled:

1'. N94K+D96L+E99K

2'. SPPRRP+N94K+D96L+T231R+N233R+D234R+Q249R

3'. SPPRRP+Al9C+C36A+N94K+D96L+Q249R

4'. STPRRP+N94R

5'. SCIRR+N94K+D96L+E239C+Q249R

6'. D137G+D167G+E210V

7'. D96L+E99K+V187A

8'. SPPRRP+D57G+N94K+D96L+Q249R

9'. N94R+F95L

The following components where mixed in a microtube:

2 μl plasmid mixture (0.15 μg/μl), specific primers flanking the gene (1pmol/μl), 2 μl 2.5 mM dNTP, 2.5 mM MgCl₂, 2 μl 10*taq buffer (PerkinElmer), 0.5 μl taq enzyme in a total volume of 20 μl.

The tube was set in a Perkin Elmer 2400 thermocycler. The followingPCR-program was run: (94° C., 5 minutes) 1 cycle: (94° C., 30 seconds,70° C., 0 seconds) 99 cycles (72° C., 2 minutes, 4° C. indefinite) 1cycle

The PCR-reaction was run on a 1.5% agarose gel. A DNA-band of thespecific expected size was cut out of the agarose gel and purified usingJETsorb (from GENOMED Inc.). The purified PCR-product was cloned into aTA-vector (from Invitrogen (the original TA cloning kit). The ligatedproduct was transformed into a standard Escherichia coli strain (DH5a).

20 transformants where fully sequenced across the gene of interest.

Result

The following 20 variants were found:

1. D137G+D167G+E210V+Y213C

2. SPPRRP+D57G+N94K+D96L+Q249R

3. N94R+F95L

4. SPPRRP+D137G+D167G+E210V

5. N94K+D96L+E99K+V187A+T267I

6. D137G+D167G+E210V

7. N94K+D96L+E99K+V187A

8. D57G+N94R+F95L+Q249R

9. N94K+D96L+E99K+E210V

10. SPPRRP+A19C+C36A+N94K+D96L

11. N94R+F95L

12. D137G+D167G+E210V

13. N94K+D96L+Q249R

14. SPPRRP+Q15P+A19C+C36A+N94K+D96L

15. SPPRRP+N94K+D96L+T231R+N233R+D234R+Q249R

16. D137G+D167G+E210V

17. SCIRR+N94K+D96L+Q249R

18. N94K+D96L+E99K

19. N94R+F95L

20. SPPRRP+N94R+F95L+F113S+Q249R

Nearly all mutations where represented (19 of 20) indicating little biasfor specific templates.

    ______________________________________                                        Statistics:                                                                   ______________________________________                                        Not shuffled           10                                                     Shuffled between at least 2 templates                                                                8                                                      Shuffled between at least 3 templates                                                                2                                                      ______________________________________                                    

The shuffled sequences can then be subcloned from the E. coli TA vectorinto the yeast vector pJSO26 as a BamHI-XbaI fragment (see WO 97/07205),and e.g. screened for new shuffled sequences with improved performancein detergents (see WO 97/07205).

Example 2

Method 2

PCR products of 10 different lipase variant genes were generated asabove and pooled in equimolar amounts.

The following 10 mutant genes were shuffled.

1'. D137G+D167G+E210V

2'. D96L+E99K+V187A

3'. N94K+D96L+E99K

4'. SPPRRP+D57G+N94K+D96L+Q249R

5'. D111N+F211A+G225P

6'. SPPRRP+N94K+D96L+T231R+N233R+D234R+Q249R

7'. SPPRRP+A19C+C36A+N94K+D96L+Q249R

8'. STPRRP+N94R

9'. N94R+F95L

10'. SCIRR+N94K+D96L+E239C+Q249R

The following mixture was generated in a suitable tube:

1 μl PCR mixture (0.1 μg), decamer random primer (300 pmol), 2 μl10*Klenow buffer (Promega), 0.25 mM dNTP, 2.5 mM MgCl₂ in a total volumeof 20 μl.

The mixture was set in a PE2400 thermocycler where the following programwas run: 96° C., 5 minutes, 25° C. 5 minutes, 0.5 ml Klenow enzyme wasadded, 25° C. 60 minutes, 35° C. 90 minutes.

This procedure generated a high number of small DNA polymers originatingfrom all parts of the gene

10 μl was taken out for test on agarose gel.

10 μl PCR mixture (0.25 mM dNTP, 1 μl 10*Taq buffer (Perkin Elmer), 2.5mM MgCl₂, 0.5 μl Taq enzyme) was added to the 10 μl in the tube in thethermocycler. Then the following standard PCR-program was run: (94° C.,5 minutes) 1 cycle, (94° C. 30 seconds, 45° C., 30 seconds, 72° C. 30seconds) 25 cycles, 72° C. 7 minutes, 4° C. indefinite.

The PCR products were run on a 1.5 agarose gel. A clear unbiased smearwas seen. DNA between 400 and 800 bp was isolated from the gel.

Half of the purified PCR product was mixed in a tube with two specificprimers (40 pmol) flanking the gene of interest, 0.25 mM dNTP, 2 μl10*Taq buffer, 2.5 mM MgCl₂. Then the following standard PCR-program wasrun: (94° C., 5 minutes) 1 cycle, (94° C. 30 seconds, 50° C., 30seconds, 72° C. 30 seconds) 25 cycles, 72° C. 7 minutes, 4° C.indefinite.

The PCR product was run on a 1.5% agarose gel. A specific though weakband of the expected size was isolated. Additional PCR was run usingspecific primers (as mentioned above) in order to amplify thePCR-product before cloning.

The PCR-product and the desired vector were cut with the appropriaterestriction enzymes (BamHI/XhoI). The vector and the PCR product wererun on a 1.5% agarose gel, and purified from the gel.

The cut PCR-product and the cut vector were mixed in a ligase bufferwith T4 DNA ligase (Promega). After overnight ligation at 16° C. themixture was transformed into E. coli strain DH5a.

19 clones were fully sequenced across the gene.

Result

The following 19 variants were found:

1. STPRRP+N94R+N233R+D234R+Q249R

2. SPPRRP+N233R+D234R+Q249R

3. D96L+D167G+Q249R

4. N94R

5. D167G

6. SPPRRP+A19C+A28T+N94K+D96L+D111N+E239C

7. SPPRRP+A19C+C36A+N94K+D96L+Q249R

8. N94K+D96L

9. N25T+D57G+N94R+E99K+D167G+T231R+N233R+D234R+Q249R

10. N94R+Q249R

11. D167G

12. D167G

13. T32I+N94R+F95L+D167G+Q249R

14. E87K+N94K+D96L

15. N94R+F95L+Q249R

16. N94K+D96L+D111N

17. STPRRP+S17T

18. N94K+D96L+V187A

19. SPPRRP+D57G+N94K+D96L+D111N+L151S

All template variants were represented indicating little bias forspecific templates.

There were no apparent hot spots with regard to mutation exchange and itseems to be evenly distributed along the gene

    ______________________________________                                        Statistics:                                                                   ______________________________________                                        Not shuffled           1                                                      Shuffled between at least 2 templates                                                                10                                                     Shuffled between at least 3 templates                                                                6                                                      Shuffled between at least 4 templates                                                                1                                                      Shuffled between at least 5 templates                                                                0                                                      Shuffled between at least 6 templates                                                                1                                                      ______________________________________                                    

The shuffled sequences can then be subcloned from the E. coli TA vectorinto the yeast vector pJSO26 as a BamHI-XbaI fragment (see WO 97/07205),and e.g. screened for new shuffled sequences with improved performancein detergents (see WO 97/07205).

Example 3

Amylase variant shuffling

In Example 1, it was shown how a number of multiple variants of H.lanuginosa lipase were shuffled. In a similar manner, variants ofBacillus α-amylases can be shuffled.

Earlier patent applications have identified variants of variousα-amylases from Bacillus species improved for particular properties,e.g. thermostability, stability under Calcium-depleted conditions,improved wash-performance etc. (see WO95/10603, WO96/23874, WO96/23873,and PCT/DK97/00197).

Variants of B. licheniformis α-amylase amyL can be shuffled as follows.The variants are all located in the B. subtilis expression vectorpDN1528 described in WO95/10603.

The experiment is carried out under the exact same conditions as Example1 except that the flanking 27mer primers used to initiate DNA synthesiswere different.

The PCR amplified band of approximately 1500 bp is purified from anagarose gel and cloned as described in Example 1. Alternativelyrestriction sites located within the amyL gene can be utilized to clonethe library of shuffled genes into either Bacillus plasmid pDN1528 or anE. coli vector containing the wild type amyL gene, e.g. pJeEN1 describedin WO96/23874

Example 4

Shuffling of two genes encoding homologous α-amylases: amyL and theamylase identified by SEQ.ID no2 (amino acid) and SEQ.ID no. 5 (DNA)described in WO96/23873.

The forward strand (identical to the mRNA) of amyL can be amplified in aPCR using standard conditions.

The forward strand is separated from the reverse strand based on itsaffinity to streptavidin coated magnetic beads and denaturation of thetwo strands with NaOH.

Similarly the reverse strand (complementary to mRNA) of the amylaseencoded by SEQ.ID no5 (WO96/23873) can be amplified and isolated

Two primer strands are used as templates in a PCR: (94° C. 5 minutes)+99cycles of (94° C., 30 seconds; 60° C., 0 seconds)+(72° C., 5 minutes)using random primers of various lengths, Taq polymerase and standardbuffer conditions as described in Example 1.

The resulting approximately 1500 bp product is cloned either as TAcloning as described in Example one (for verification of the sequence ofthe resulting clone) or into Bacillus vector, pTVB110 utilizing SfiI andPstI restriction sites.

The original template can be removed from the PCR at any step (e.g.after 5, 10 or 20 cycles) based on the biotin tag).

What is claimed is:
 1. A method of shuffling polynucleotides,comprising(a) providing an isolated sense strand from at least a firstvariant form of a polynucleotide, and an isolated antisense strand fromat least a second variant form of the polynucleotide, wherein theisolated sense and antisense strands are starting substrates to beshuffled; and (b) shuffling the isolated sense and antisense strandsprovided in step (a) by contacting the strands with at least one primeror one pair of primers and conducting a multi-cyclic polynucleotideextension reaction wherein(i) in at least one cycle, the primer(s)anneal to the isolated strands and generate extended primers; and (ii)in at least one subsequent cycle, the extended primers generated in aprevious cycle act as both primers and templates to form furtherextended primers, whereby the polynucleotide extension is continued forsufficient cycles until the further extended primers includesrecombinant forms of the shuffled polynucleotides.
 2. The method ofclaim 1, wherein the isolated strands are labeled with biotin.
 3. Themethod of claim 1, wherein each sense and antisense strand comprises apool of diverse forms of a polynucleotide to be shuffled.
 4. The methodof claim 1, wherein the first and second forms of the polynucleotidevariants exhibit more than 50% sequence identity.
 5. The method of claim4, wherein the polynucleotide variants exhibit more than 70% sequenceidentity.
 6. The method of claim 5, wherein the polynucleotide variantsexhibit more than 90% sequence identity.
 7. The method of claim 6,wherein the polynucleotide variants exhibit more than 95% sequenceidentity.
 8. The method of claim 1, wherein the at least one primer iscompletely random.
 9. The method of claim 1, wherein the at least oneprimer or at least a pair of primers is (are) partly random.
 10. Amethod of identifying polypeptides exhibiting a desired property,comprising the method of claim 1, further comprising step (c) expressingand screening a polypeptide encoded by the shuffled polynucleotide for adesired property.
 11. The method of claim 10, wherein the property is anenzymatic activity.
 12. The method of claim 1, wherein the at leastfirst and second variants forms are from naturally occurring organismsof different species.
 13. The method of claim 1, further comprisingseparating the extended primers from the starting substrates.
 14. Themethod of claim 1, wherein at least one extension cycle is conductedunder conditions of incomplete elongation.
 15. The method of claim 1,further comprising selecting the starting substrates to be shuffled. 16.A method for shuffling polynucleotides, comprising(a) providing at leasttwo variant forms of a polynucleotide as starting substrates to beshuffled; and (b) shuffling the at least two variant polynucleotides bycontacting the variants with a population of completely or partiallyrandom primers; and conducting a multi-cyclic polynucleotide extensionreaction wherein(i) in at least one cycle, the primers anneal to thevariants to generate extended primers, wherein the at least one cycle isconducted with a shorter extension time than required for completeextension of the starting substrates; and (ii) in at least onesubsequent cycle, the extended primers generated in a previous cycle aredenatured to single-stranded fragments, which anneal in new combinationsforming annealed fragments, whereby one strand of an annealed fragmentprimes replication of the other to form further extended primers;whereby the polynucleotide extension is continued for sufficient cyclesuntil the further extended primers includes recombinant forms of theshuffled polynucleotide.
 17. The method of claim 16, wherein thestarting substrates are labeled, and separated from the extended primersgenerated in step (b) by use of the label.
 18. The method of claim 16,wherein the multi-cyclic polynucleotide extension reaction is conductedunder conditions promoting misincorporation of nucleotides.
 19. Themethod of claim 16, wherein the starting substrates have a length of 50bp to 20 kb.
 20. The method of claim 16, wherein the multi-cyclicpolynucleotide extension reaction is conducted with a thermostable DNApolymerase.
 21. The method of claim 20, wherein the polymerase is Taq,Amplitaq, Vent, or Pwo.
 22. The method of claim 16, wherein themulti-cyclic polynucleotide extension reaction is conducted with apolymerase selected from the group consisting of T4 polymerase, T7polymerase, E. coli DNA polymerase I and the Klenow fragment of DNApolymerase I, and wherein said polymerase is added to the reactionmixture after each cycle.
 23. The method of claim 16, wherein themulti-cyclic polynucleotide extension reaction is conducted in thepresence of an RNA polymerase.
 24. The method of claim 16, wherein saidpolymerase is an error-prone polymerase.
 25. The method of claim 16,wherein the starting substrates are natural variants.
 26. The method ofclaim 16, wherein at least two starting substrates exhibit more than 50%sequence identity.
 27. The method of claim 26, wherein thepolynucleotide variants exhibit more than 70% sequence identity.
 28. Themethod of claim 27, wherein the polynucleotide variants exhibit morethan 90% sequence identity.
 29. The method of claim 27, wherein thepolynucleotide variants exhibit more than 95% sequence identity.
 30. Themethod of claim 16, wherein the primers are labeled.
 31. The method ofclaim 30, wherein the label is biotin or digoxigenin.
 32. The method ofclaim 16, wherein the primers are 6 to 200 bp in length.
 33. The methodof claim 32, wherein the primers are 10 to 70 bp.
 34. The method ofclaim 33, wherein the primers are 15 to 40 bp.
 35. The method of claim16, wherein the substrates are from naturally occurring organisms ofdifferent species.
 36. The method of claim 16, wherein the startingsubstrates are linear DNA fragments generated by a PCR reaction.
 37. Themethod of claim 16, wherein the starting substrates are cloned intovectors.
 38. The method of claim 16, wherein the starting substratesencode variant forms of an enzyme.
 39. The method of claim 38, whereinenzyme is selected from the group consisting of carbonyl hydrolase,carbohydrase, an esterase, a protease, a lipase, an amylase, acellulase, an oxidase, and an oxido reductase.
 40. The method of claim16, wherein the starting substrates encode variant forms of apolypeptide selected from the group consisting of insulin, ACTH,glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone,pituary hormones, somatomedin, erythropoietin, luteinizing hormone,chorionic gonadotropin, hypothalamic releasing factors, antidiuretichormones, thyroid stimulating hormone, relaxin, interferon,thrombopoeitin (TPO) and prolactin.
 41. The method of claim 16, whereinthe starting substrates have a function selected from the groupconsisting of transcription initiation, transcription termination,translational initiation, and operator sites related to expression ofgenes.
 42. The method of claim 16, wherein the number of cycles is fewerthan 100 cycles.
 43. A method for shuffling polynucleotides,comprising(a) providing at least two variants of a polynucleotide asstarting substrates to be shuffled; and (b) shuffling the at least twovariant polynucleotides by contacting the variants with a population ofcompletely or partially random primers, and conducting a multi-cyclicpolynucleotide extension reaction wherein(i) in at least one cycle, theprimers anneal to the variant forms and primer replication of thevariants thereby generating extended primers, wherein the at least onecycle is terminated by an increase in temperature to terminateextension; and (ii) in at least one subsequent cycle, the extendedprimers generated in a previous cycle are denatured to single-strandedfragments, which anneal in new combinations forming annealed fragments,whereby one strand of an annealed fragment primes replication of theother to form further extended primers; whereby the polynucleotideextension is continued for sufficient cycles until the further extendedprimers includes recombinant forms of the shuffled polynucleotide. 44.The method of claim 43, wherein the temperature is raised to between 90°C. and 99° C.
 45. The method of claim 43, wherein the startingsubstrates are labeled, and the separated from the extended primersgenerated in step (b) by use of the label.
 46. The method of claim 43,wherein the multi-cyclic polynucleotide extension reaction is conductedunder conditions promoting misincorporation of nucleotides.
 47. Themethod of claim 43, wherein the starting substrates have a length of 50bp to 20 kb.
 48. The method of claim 43, wherein the multi-cyclicpolynucleotide extension reaction is conducted with a polymeraseselected from the group consisting of T4 polymerase, T7 polymerase, E.coli DNA polymerase I and the Klenow fragment of DNA polymerase I, andwherein said polymerase is added to the reaction mixture after eachcycle.
 49. The method of claim 43, wherein the multi-cyclicpolynucleotide extension reaction is conducted in the presence of an RNApolymerase.
 50. The method of claim 43, wherein said polymerase is anerror-prone polymerase.
 51. The method of claim 43, wherein the startingsubstrates are natural variants.
 52. The method of claim 43, wherein atleast two starting substrates exhibit more than 50% sequence identity.53. The method of claim 52, wherein at least two starting substratesexhibit more than 70% sequence identity.
 54. The method of claim 53,wherein at least two starting substrates exhibit more than 90% sequenceidentity.
 55. The method of claim 54, wherein at least two startingsubstrates exhibit more than 95% sequence identity.
 56. The method ofclaim 43, wherein the primers are labeled.
 57. The method of claim 56,wherein the primers are 10 to 70 bp.
 58. The method of claim 56, whereinthe label is biotin or digoxigenin.
 59. The method of claim 58, whereinthe primers are 15 to 40 bp.
 60. The method of claim 43, wherein theprimers are 6 to 200 bp.
 61. The method of claim 43, wherein thesubstrates are from naturally occurring organisms of different species.62. The method of claim 43, wherein the starting substrates are linearDNA fragments generated by a PCR reaction.
 63. The method of claim 43,wherein the starting substrates are cloned into vectors.
 64. The methodof claim 43, wherein the starting substrates encode variant forms of anenzyme.
 65. The method of claim 64, wherein enzyme is selected from thegroup consisting of carbonyl hydrolase, carbohydrase, an esterase, aprotease, a lipase, an amylase, a cellulase, an oxidase, and an oxidoreductase.
 66. The method of claim 43, wherein the starting substratesencode variant forms of a polypeptide selected from the group consistingof insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin,parathyroid hormone, pituary hormones, somatomedin, erythropoietin,luteinizing hormone, chorionic gonadotropin, hypothalamic releasingfactors, antidiuretic hormones, thyroid stimulating hormone, relaxin,interferon, thrombopoeitin (TPO) and prolactin.
 67. The method of claim43, wherein the starting substrates have a function selected from thegroup consisting of transcription initiation, transcription termination,translational initiation, and operator sites related to expression ofgenes.
 68. The method of claim 43, wherein the number of cycles is fewerthan 100 cycles.
 69. A method for shuffling polynucleotides,comprising:(a) providing at least two variants of a polynucleotide to beshuffled; and (b) shuffling the at least two variant polynucleotides bycontacting the variants with a population of completely or partiallyrandom primers, and conducting a multi-cyclic polynucleotide extensionreaction wherein(i) in at least one cycle, the primers anneal to thevariants and prime replication of the variants thereby generatingextended primers, wherein at least one cycle is conducted with theKlenow fragment of DNA polymerase I; and (ii) in at least one subsequentcycle, the extended primers generated in a previous cycle are denaturedin single-stranded fragments which anneal in new combinations formingannealed fragments, whereby one strand of an annealed fragment primesreplication of the other to form further extended primers; whereby thepolynucleotide extension is continued for sufficient cycles until thefurther extended primers includes recombinant forms of the shuffledpolynucleotide.
 70. The method of claim 69, wherein the startingsubstrates are labeled, and the separated from the extended primersgenerated in step (b) by use of the label.
 71. The method of claim 69,wherein the multi-cyclic polynucleotide extension reaction is conductedunder conditions promoting misincorporation of nucleotides.
 72. Themethod of claim 69, wherein the starting substrates have a length of 50bp to 20 kb.
 73. The method of claim 69, wherein the starting substratesare natural variants.
 74. The method of claim 69, wherein at least twostarting substrates exhibit more than 50% sequence identity.
 75. Themethod of claim 74, wherein at least two starting substrates exhibitmore than 70% sequence identity.
 76. The method of claim 75, wherein atleast two starting substrates exhibit more than 90% sequence identity.77. The method of claim 76, wherein at least two starting substratesexhibit more than 95% sequence identity.
 78. The method of claim 69,wherein the primers are labeled.
 79. The method of claim 78, wherein thelabel is biotin or digoxigenin.
 80. The method of claim 69, wherein theprimers are 6 to 200 bp in length.
 81. The method of claim 80, whereinsaid primers are 10 to 70 bp in length.
 82. The method of claim 81,wherein said primers are 15 to 40 bp in length.
 83. The method of claim69, wherein the substrates are from naturally occurring organisms ofdifferent species.
 84. The method of claim 69, wherein the startingsubstrates are linear DNA fragments generated by a PCR reaction.
 85. Themethod of claim 69, wherein the starting substrates are cloned intovectors.
 86. The method of claim 69, wherein the starting substratesencode variant forms of an enzyme.
 87. The method of claim 86, whereinenzyme is selected from the group consisting of carbonyl hydrolase,carbohydrase, an esterase, a protease, a lipase, an amylase, acellulase, an oxidase, and an oxido reductase.
 88. The method of claim69, wherein the starting substrates encode variant forms of apolypeptide selected from the group consisting of insulin, ACTH,glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone,pituary hormones, somatomedin, erythropoietin, luteinizing hormone,chorionic gonadotropin, hypothalamic releasing factors, antidiuretichormones, thyroid stimulating hormone, relaxin, interferon,thrombopoeitin (TPO) and prolactin.
 89. The method of claim 69, whereinthe starting substrates have a function selected from the groupconsisting of transcription initiation, transcription termination,translational initiation, and operator sites related to expression ofgenes.
 90. The method of claim 69, wherein the number of cycles is fewerthan 100 cycles.
 91. A method of shuffling polynucleotides,comprising:shuffling first strands of a population of polynucleotideswithout shuffling second strands; and synthesizing strands complementaryto the shuffled first strands to form shuffled duplex polynucleotides.92. The method of claim 91, wherein the first strands are isolated fromthe second strands by labeling the with biotin.
 93. The method of claim91, wherein the first strands are a pool of polynucleotides comprisingdiverse forms of a polynucleotide.
 94. The method of claim 93, whereinthe diverse forms of the polynucleotide are from naturally occurringorganisms of different species.
 95. The method of claim 91, wherein thepool of polynucleotides exhibit more than 50% sequence identity.
 96. Themethod of claim 95, wherein the pool of polynucleotides exhibit morethan 70% sequence identity.
 97. The method of claim 96, wherein the poolof polynucleotides exhibit more than 90% sequence identity.
 98. Themethod of claim 97, wherein the pool of polynucleotides exhibit morethan 95% sequence identity.
 99. The method of claim 91, wherein thefirst strands are contacted with at least one completely random primer.100. The method of claim 91, wherein the first strands are contactedwith at least one partly random primer or at least a pair of partlyrandom primers.
 101. A method of identifying polypeptides exhibiting adesired property, comprising the method of claim 91, further comprisingexpressing and screening a polypeptide encoded by the shuffledpolynucleotide for a desired property.
 102. The method of claim 101,wherein the property is an enzymatic activity.
 103. The method of claim91, wherein at least one extension cycle is conducted under conditionsof incomplete elongation.
 104. The method of claim 91, wherein thepopulation of polynucleotides encode variant forms of an enzyme. 105.The method of claim 104, wherein enzyme is selected from the groupconsisting of carbonyl hydrolase, carbohydrase, an esterase, a protease,a lipase, an amylase, a cellulase, an oxidase, and an oxido reductase.106. The method of claim 105, wherein the population of polynucleotidescomprises at least two variant polynucleotides and wherein the variantpolynucleotides encode a polypeptide selected from the group consistingof insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin,parathyroid hormone, pituary hormones, somatomedin, erythropoietin,luteinizing hormone, chorionic gonadotropin, hypothalamic releasingfactors, antidiuretic hormones, thyroid stimulating hormone, relaxin,interferon, thrombopoeitin (TPO) and prolactin.
 107. In a method ofshuffling polynucleotides from variants by template directedpolynucleotide extension of a population of fragments ofpolynucleotides, the improvement wherein opposing strands ofpolynucleotides to be shuffled are separated before conducting theshuffling step, and the shuffling reaction is conducted in the presenceof only one strand from each variant.
 108. The method of claim 107,wherein the opposing strands are separated by labeling with biotin. 109.The method of claim 107, wherein the polynucleotides to be shuffledexhibit more than 50% sequence identity.
 110. The method of claim 109,wherein the polynucleotides to be shuffled exhibit more than 70%sequence identity.
 111. The method of claim 110, wherein thepolynucleotides to be shuffled exhibit more than 90% sequence identity.112. The method of claim 111, wherein the polynucleotides to be shuffledexhibit more than 95% sequence identity.
 113. The method of claim 107,wherein the strands to be shuffled are contacted with at least onecompletely random primer.
 114. The method of claim 107, wherein thestrands to be shuffled are contacted with at least one partly randomprimer or at least a pair of partly random primers.
 115. The method ofclaim 107, wherein the strands to be shuffled are contacted with atleast one primer 6 to 200 bp in length.
 116. The method of claim 115,wherein the strands to be shuffled are contacted with at least oneprimer 10 to 70 bp in length.
 117. The method of claim 116, wherein thestrands to be shuffled are contacted with at least one primer 15 to 40bp in length.
 118. A method of identifying polypeptides exhibiting adesired property, comprising the method of claim 107, further comprisingexpressing and screening a polypeptide encoded by the shuffledpolynucleotide for a desired property.
 119. The method of claim 118,wherein the property is an enzymatic activity.
 120. The method of claim107, wherein the variant polynucleotides are from naturally occurringorganisms of different species.
 121. The method of claim 107, whereinthe shuffling reaction is performed With at least one extension cycleconducted under conditions of incomplete elongation.
 122. The method ofclaim 107, wherein the population of polynucleotides encode diverseforms of an enzyme.
 123. The method of claim 122, wherein enzyme isselected from the group consisting of carbonyl hydrolase, carbohydrase,an esterase, a protease, a lipase, an amylase, a cellulase, an oxidase,and an oxido reductase.
 124. The method of claim 107, wherein thepopulation of polynucleotides comprises at least two variantpolynucleotides and wherein the variant polynucleotides encode apolypeptide selected from the group consisting of insulin, ACTH,glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone,pituary hormones, somatomedin, erythropoietin, luteinizing hormone,chorionic gonadotropin, hypothalamic releasing factors, antidiuretichormones, thyroid stimulating hormone, relaxin, interferon,thrombopoeitin (TPO) and prolactin.
 125. A method for shufflingpolynucleotides, comprising:(a) isolating a sense strand from an atleast one first polynucleotide variant and an antisense strand from anat least one second polynucleotide variant, wherein only the isolatedstrands are templates for shuffling; (b) shuffling the isolated strandsof the polynucleotide variants by contacting the variant polynucleotideswith a population of primers and conducting a multi-cyclicpolynucleotide extension to form shuffled double-strandedpolynucleotides, wherein at least one cycle is conducted with a shorterextension time than required for complete extension of the templates.126. A method for shuffling polynucleotides, comprising:(a) isolating asense strand from an at least one first polynucleotide variant and anantisense strand from at least one second polynucleotide variant,wherein only the isolated strands are templates for shuffling; (b)shuffling the isolated strands of the polynucleotide variants bycontacting the variants with a population of primers and conducting amulti-cyclic polynucleotide extension to form shuffled double-strandedpolynucleotides, wherein at least one cycle is terminated by an increasein temperature to terminate extension.